Protein phosphatase 2A (PP2A) is an abundant serine/threonine-specific phosphatase, which plays an important role in many fundamental processes, including development, cell division, and cancer. It exists in cells in two forms, the core enzyme, composed of the catalytic C subunit and a regulatory A subunit, and the holoenzyme, consisting of core enzyme to which one of several B subunits is bound. B subunits are the key regulators of PP2A. They fall into three families, designated B, B', and B", which are unrelated by protein sequence. Tumor (T) antigens encoded by polyoma viruses promote cell proliferation by binding to the core enzyme. Work in this laboratory has focused on the structure and function of PP2A. A model of PP2A subunit and T antigen interaction was developed based on data from site-directed mutagenesis of the A subunit. In addition, it was demonstrated that PP2A is required for initiation of DNA replication but not for elongation of previously engaged replication forks. The hypothesis has been proposed that protein kinases not only activate initiation, as well known, but also inactivate this process, and that the inactivating kinase(s) are counteracted by PP2A. The specific aims of the proposal are: 1. To investigate the precise role of PP2A in DNA replication. The main focus will be on identifying the substrate(s) of PP2A. Potential candidates are ORC, cdc6, MCM, cdc45, RPA, and DNA polymerase alpha, known to be involved in initiation. Other potential substrates are the protein kinases cdk2-cyclin E and cdc7/dbf4 which activate the initiation complex. This work will be carried out using a cell- free DNA replication system derived from Xenopus leavis eggs, in which sperm chromatin is first incubated with egg cytosol to allow formation of a pre-replication complex. Subsequent addition of a nucleoplasmic extract leads to a complete round of replication. 2. To continue site-directed mutagenesis of the A subunit in order to generate additional mutants defective in binding specific regulatory B subunits and T antigens. The A subunit mutants will be assayed by co-immunoprecipitation for binding B subunits and T antigens. In addition, the binding properties of the mutants will be tested in vivo by mixed infection of Sf9 cells with recombinant baculoviruses encoding the mutant A subunit, C subunit, and B subunit or T antigen, and by measuring formation of core enzyme and holoenzyme. The mutants generated will be used as tools in the future for elucidating the role of PP2A and of its interaction with T antigens in cancer.